Contents of Volume 53

Plant Molecular Biology -     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Eye size variation reflects habitat and daily activity patterns in colubrid snakes

The functioning of the vertebrate eye depends on its absolute size, which is presumably adapted to specific needs. Eye size variation in lidless and spectacled colubrid snakes was investigated, including 839 specimens belonging to 49 genera, 66 species and subspecies. Variations of adult eye diameters (EDs) in both absolute and relative terms between species were correlated with parameters reflecting behavioral ecology. In absolute terms, eye of arboreal species was larger than in terrestrial and semiaquatic species. For diurnal species, EDs of terrestrial species do not differ from semiaquatic species; for nocturnal species the ED of terrestrial species is larger than fossorial species but not different from semiaquatic species. In relative terms, ED did not differ significantly by habitat for diurnal species. Although the ED of terrestrial species is larger than fossorial species there were no differences for nocturnal species between semiaquatic and fossorial snakes. In contrast to other vertebrates studied to date, colubrid EDs in absolute and relative terms are larger in diurnal than in nocturnal species. These observations suggest that among colubrid snakes, eye size variation reflects adaptation to specific habitats, foraging strategies and daily activities, independently of phylogeny. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Winter food availability limits winter survival of Mongolian gerbils (<Emphasis Type="Italic">Meriones unguiculatus</Emphasis>)

Food availability is important to the dynamics of animal social organizations or populations. However, the role of winter food availability in animal population dynamics is still controversial. We carried out an experimental study to test Lack’s hypothesis that reduced food in winter limits survival and spring numbers of breeding individuals of social groups, using the Mongolian gerbil (Meriones unguiculatus) as model species. We established 24 gerbil social groups in 24, 10 × 10 m, pens in September 2008. We provided wheat seeds as supplemental food in 12 enclosures from September 2008 to March 2009; the other 12 enclosures, not provided with supplemental food, served as controls. We live-trapped gerbils at a 2-week interval from September to April. Supplemental food during winter increased biweekly survival by 10% relative to that in control groups. Only four control social groups survived to the end of our study whereas all 12 food-supplemented social groups survived through our study period. Supplemental food also increased cumulative numbers of recruits and group sizes of gerbils. We conclude that winter food availability limits winter survival and spring social groups or population sizes of Mongolian gerbils.     

The distribution of carbohydrate side chains along the polypeptide chain of glucoamylase

Glucoamylase is a starch-hydrolyzing enzyme with a glycoprotein structure, used industrially for the conversion of starch to glucose, citric acid, corn syrups, and high-fructose sweeteners. This enzyme possesses an unusual type of structure in which many carbohydrate side chains are linked O-glycosidically to serine and threonine residues of the polypeptide chain. The carbohydrate side chains may be single monosaccharide residues or oligosaccharides of mannose, glucose, galactose, and in some cases N-acetylglucosamine. New data from experiments on the CNBr fragmentation of glucoamylase followed by chemical and immunological characterization of the fragments show that the carbohydrate side chains are distributed randomly along the polypeptide chain. Such a structure is appropriately termed a random model reprensentation for the glucoamylase molecule.     

Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM alphaW62A,betaW120A (DM(W62A/W120A)) double mutant was expressed in HLA-DR(+) HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.